Modification of alginates or other uronic acid compounds by treatment with CO2

ABSTRACT

A method for modification of alginates or other uronic acid containing compounds to give improved physical properties, wherein the alginates in solid form are treated with CO 2  under supercritical conditions thereby achieving at least partial epimerization of said uronic acid containing compound. The critical point of CO 2  is pcrit=73.83 bar and tcrit=31° C.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for modification of alginatesand other uronic acid substances to obtain changes in the functionalproperties, in particular with respect to the ability of forming gelswith inorganic or organic polyvalentions.

2. Prior Art

Alginates are manufactured from brown sea-weed and are utilized inseveral applications where their polyelectrolytic nature forms the basisfor e.g. gelation, thickening as well as water- and ion-binding.

Chemically speaking, alginates constitute a group of linear, binarycopolymers built up of salts of β-D-mannuronic acid (M) and its C-5epimer, α-L-guluronic acid (G). The M and G units are found in threetypes of sequences; G-rich sequences called G-blocks, M-rich sequencescalled M-blocks, and alternating sequences found in MG-blocks,symbolized MGMG. The fractional content of these monomer units as wellas their sequencial distribution varies with the algal source. The ionbinding and gelforming properties depend on the monomer fractions, butin particular on the distribution of G-units along the chain. A highcontent of G-blocks leads to the technically important gel-formingproperties.

SUMMARY OF THE INVENTION

The object of the present invention is to convert M-units to G-units inoligomers or polymers, in order to change their physical properties.This type of endo-epimerisation of the intact polymer may be performedby treating alginate with mannuronan C-5 epimerase, an enzymeparticipating in the in vivo biosynthesis of alginate (cf. NOapplication 845059).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 generally shows a 400 MHz ¹ H n.m.r. spectra of Na-alginate fromL. hyperborea fronds. Specifically, (A) shows Na-alginate from L.hyperborea fronds, treated with CO₂ under supercitical conditions for 12hours at 500 bar and 45° C. and (B) shows untreated alginate. The changein G content of the modified polymer is demostrated by the relativeintensity increase in "G signals" (I and III) compared to "M signals"(II).

FIG. 2 generally shows a 400 MHz ¹ H n.m.r. spectra of sodiumpolymannuronated isolated from A. nodosum. Specifically, (A) showsNa-ploy-M from A. nodosum, treated with CO₂ under superoriticalconditions for 12 hours at 200 bar (F_(G) =0.16) and (B) shows untreatedpoly-M (F_(G) <0.05). Signal I in the spectrum corresponds to theL-guluronate content.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention such epimerisation, well known as anenzymatic process from several polysaccharide producing organisms, mayalso be performed without enzymes. This kind of process has previouslynot been possible on a polymer level, as an alkali catalyzed abstractionof H-5, resulting in a carbanion (charge at C-5), will lead tohydrolysis ot the polymer chain in the presence of water. Furthermore,most polyuronides (.e.g. alginate) are insoluble in non-polar solvents,reactions in aprotic solvents will therefore be difficult to perform.

The present invention is based on a completely new principle thateliminates the problem mentioned above by treating alginate in the solidstate with carbon dioxide under supercritical conditions, where thedensity is like in a liquid, but the diffusion rate is as in the gasstate. CO₂ may then act both as an aprotic solvent and as a catalyst inthe alkaline removal of H-5, resulting in a C-5 carbanion. The highpressure will contribute energetically to the epimerisation, as thediaxially linked G-units give a more compact chain structure, and hence,a lower partial molar volume as compared to the diequatorially linkedM-units. Furthermore, in aprotic solvents the anomeric effect rendersaxial substitution in C-1 more favorable, as in α-L-guluronate inalginate.

Carbon Dioxide has a critical point, defined by a specific pressure ofCO₂ Pcrit=73.83 bar and a temperature of CO2 tcrit =31° C.

As starting material alginate from algal or bacterial sources may bechosen, either as salts (alkali or alkaline earth metal forms), estersor other acid derivatives.

Applications of supercritical carbon dioxide are hitherto found mainlywithin extraction of e.g. aroma compounds, using the supercritical fluidas an inert solvent (see H. Coenen and E. Kriegel, "Uses ofSupercritical Gas Extraction in the Food Industry", J. Chem. Ing. Tech.55 (1983) 890). The invention forming the basis for the presentinvention is the first known example of a non-enzymatic, chemicalepimerization of polysaccharides, and the first application of asupercritical fluid as both catalyst and solvent in a chemical reaction.The present invention also differs from enzymatic and most other knownchemical modification processes of this kind in being applicable to asolid state material. The known processes mentioned require dilutesolutions (<1%), and the product must be precipitated, purified anddried in a costly process.

We have treated alginate with CO₂ under supercritical conditions usingvarying pressures (150-500 bar), and the experimental conditions werereflected in a varying, but significant epimerisation, a correspondingincrease in gel strength also accompanied the epimerisation. Thus, theresult suggests that C-5 epimerisation of glycuronans or correspondingoligosaccharides by supercritical carbon dioxide treatment provides acompletely new method for chemical modification of carbohydrates.

The chemical process being the object of the present invention has agreat economic potential due to the following:

(A) It may be performed on solid state material.

(B) No toxic or hazardous chemicals are needed, and hence, nopurification of the product is required.

(C) The process may conveniently be performed on a large scale astechnology for extraction with supercritical fluids is already developedfor other purposes.

(D) The process is not limited to epimerisation of β-D-mannuronateresidues in alginate, but may as well be utilized for epimerisation ofother polyuronides as e.g. C-6 oxidized cellulose, where D-glucuronicacid is converted to its C-5 epimer L-iduronic acid. Correspondingreactions may be utilized in treatment of mucopolysaccharides, e.g.chondroitin sulfate and hyaluronic acid:

EXAMPLE 1

Sodium alginate (50 g) isolated from Laminaria hyperborea was packed ina steel column and treated with CO₂ under supercritical conditions. Thepressure was varied between 150 and 500 bar. The gas flow was 0.5-10ml/min, the temperature 45° C. and the treatment lasted for 12 hours.Loss of material was <1% in all experiments. The polymer composition wasdetermined by high-field (400 MHz) ¹ H NMR spectroscopy (cf. FIG. 1) (H.Grasdalen "High-field ¹ H-NMR Spectroscopy of Alginate: SequentialStructure and Linkage Conformation." Carbohydr. Res. 118 (1983) 255-260)and is expressed in Table I as the molar fractions of the two monomerunits, M and G, as F_(M) and F_(G), repectively, where F_(M) +F_(G) =1,and as the sequence parameters or fractions of the four "Nearestneighbour" sequences, MM, GG, MG, and GM, where F_(MM) +F_(GG) +2F_(MG)=1.

The results in table I show that CO₂ treatment has resulted in asignificant increase in the guluronate content by three differentpressure conditions. The content, as expressed by F_(G), changed from0.52 (i.e. 52%) in the untreated material to 0.60, 0.64 and 0.66 afterthe treatment. The highest pressure (500 bar) gave the greatest changein composition.

This increase in G content resulted in alginates with an increasedability to form gels with polyvalent ions. Table II shows some physicalproperties of the CO₂ treated polymers. Viscosities were determined in aBrookfield viscometer and intrinsic viscosities [η] were determined in aCannon-Ubbelohde capillary viscosimeter. Gel strengths of 1% calciumalginate gels were determined in a FIRA gel strength apparatus, and themodulus of rigidity in 2% homogenous calcium gels were determined in aStevens Texture Analyser (G. Skjåk-Br K, B. Larsen and O. Smidsr d"Tailoring of Alginate By Enzymatic Modification in vitro." Int. J.Biol. Macromol. 8 (1986) 330-336).

The CO₂ treatment resulted in a decrease in the intrinsic viscosity,supposedly caused by hydrolytic cleavage of glycosidic bonds. However,the limited degradation has no influence on the gel forming propertiesof alginates. The gel strength, in terms of both the FIRA values and themodulus of rigidity, showed a pronounced increase and let to valuescomparable to those found for the most strongly gel-forming alginatesobtained from the stipe of Laminaria hyperborea.

                  TABLE I                                                         ______________________________________                                        MONOMER COMPOSITION AND SEQUENCE                                              PARAMETERS FOR CO.sub.2 -TREATED ALGINATE.                                            F.sub.G F.sub.M                                                                              F.sub.GG  F.sub.GM                                                                           F.sub.MM                                ______________________________________                                        Untreated 0.52      0.48   0.34    0.18 0.30                                  sample                                                                        150 bar   0.60      0.40   0.50    0.10 0.30                                  250 bar   0.64      0.36   0.55    0.09 0.27                                  500 bar   0.66      0.34   0.53    0.13 0.21                                  ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        PHYSICAL PROPERTIES OF CO.sub.2 -TREATED ALGINATE                                                Gel strength                                                                                  Modulus of                                                          FIRA      rigidity                                   Viscosity in  Intrinsic vis-                                                                           (ml)      (N/cm.sup.2)                               water (1%)    cosity [η]                                                                           1% calcium                                                                              2% calcium                                 (mPa · s)                                                                          (dl/g)     gel       gel                                        ______________________________________                                        Untreated                                                                             205       8.8        40      6.2                                      sample                                                                        150 bar 80.6      6.9        63      --                                       250 bar 86.2      6.8        63.5    --                                       500 bar 80.7      6.9        63.5    9.0                                      ______________________________________                                    

EXAMPLE 2

The sodium salt of poly-mannuronic acid isolated from Ascophyllumnodosum was treated for 12 hours with supercritical CO₂ at 200 bar and45° C. The loss was <0.5%. The guluronate content in the polymerincreased from <5% to 16% (cf. FIG. 2). The result clearly suggests thatD-mannuronate of the intact polymer is C-5 epimerised under the giveconditions.

We claim:
 1. A method for producing at least partial epimerization of auronic acid containing compound, comprising the steps of:providing auronic acid containing compound in solid form; and treating said uronicacid containing compound with CO₂ under supercritical conditions therebyachieving at least partial epimerization of said uronic acid containingcompound, wherein the critical point of CO₂ is pcrit=73.83 bar andtcrit=31° C.
 2. A method according to claim 1, wherein the uronic acidcontaining compound is an alginate.
 3. A method according to claim 1,wherein the uronic acid containing compound is a member selected fromthe group consisting of salts, acids and esters of a uronic acid.
 4. Amethod according to claim 1, wherein the uronic acid containing compoundis an alginic acid derivative.
 5. A method according to claim 1, whereinsaid uronic acid containing compound is treated with CO₂ in a batch-wisemanner.
 6. A method according to claim 1, wherein said uronic acidcontaining compound is treated with CO₂ by continuous flushing.
 7. Amethod according to claim 1, wherein the uronic acid containing compoundis selected from the group consisting of monouronides, oligouronides,polyuronides and other uronate containing polymers.
 8. A methodaccording to claim 1, wherein the uronic acid containing compound isheparin.
 9. A method according to claim 1, wherein the uronic acidcontaining compound is chondroitin sulfate.